Production of chemicals via synthetic enzymatic pathways in microbial hosts has proven useful for many important classes of molecules, including isoprenoids, polyketides, nonribosomal peptides, bioplastics, and chemical building blocks. Due to the inherent modularity of biological information, synthetic biology holds great potential for expanding this list of microbially produced compounds even further. Yet embedding a novel biochemical pathway in the metabolic network of a host cell can disrupt the subtle regulatory mechanisms that the cell has evolved over the millennia. Indeed, the final yield of a compound is often limited by deleterious effects on the engineered cell's metabolism that are difficult to predict due to the limited understanding of the complex interactions that occur within the cell. The unregulated consumption of cellular resources, metabolic burden of heterologous protein production, and accumulation of pathway intermediates/products that are inhibitory or toxic to the host are all significant issues that may limit overall yield.
The concept of metabolic engineering has emerged to fulfill this purpose, which can be defined as purposeful modification of metabolic and cellular networks by employing various experimental techniques to achieve desired goals. What distinguishes metabolic engineering from genetic engineering and strain improvement is that it considers metabolic and other cellular network as a whole to identify targets to be engineered. In this sense, metabolic flux is an essential concept in the practice of metabolic engineering. Although gene expression levels and the concentrations of proteins and metabolites in the cell can provide clues to the status of the metabolic network, they have inherent limitations in fully describing the cellular phenotype due to the lack of information on the correlations among these cellular components. Metabolic fluxes represent the reaction rates in metabolic pathways, and serve to integrate these factors through a mathematical framework. Thus, metabolic fluxes can be considered as one way of representing the phenotype of the cell as a result of interplays among various cell components; the observed metabolic flux profiles reflect the consequences of interconnected transcription, translation, and enzyme reactions incorporating complex regulations.
Cell-free synthesis offers advantages over in vivo production methods. Importantly, the lack of a cell wall in vitro allows for direct control of the synthesis environment. The redox potential, pH, or ionic strength can be altered directly, and over more broad, non-physiological ranges, due to lack of cell growth or need for cell viability. Furthermore, direct recovery of products can more easily be achieved. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production from one pathway. The present compositions and methods address these issues.